Tumors are comprised of a variety of cell types in addition to the malignant cells, including stroma, vasculature, and infiltrating immune cells. An understanding of the identities, numbers, and states of these other cellular components is clearly important to understanding the biology of tumors, and their responses to therapy, as outlined in the NCI Provocative Question C4. Recently, immune-modulatory therapies and adoptive cell therapies have become established as powerful therapeutic approaches in cancer, raising a critical need to be able to monitor immune responses directly within tumors. Positron emission tomography (PET) provides highly sensitive, non-invasive and quantitative imaging, which can be combined with antibody-based probes to provide molecular imaging of tissues and cells in vivo based on cell surface phenotype. Furthermore, engineered antibody fragments (such as cys-diabodies; covalent dimers of single-chain antibody fragments) have been generated with optimal kinetics enabling rapid in vivo targeting and clearance for imaging applications by immunoPET. In the proposed work, immunoPET using engineered antibody fragments that recognize CD8, a classic cell surface marker on cytotoxic T cells, will be developed to address the challenge of direct imaging of immune cell subsets in tumors. Specific Aim 1 will focus on the need for imaging agents to be biologically inert, and will assess biological effects (activation, cytokine release) of treating T cells with anti- CD8 cys-diabodies,in vitro and in vivo. Specific Aim 2 will employ radiolabeled anti-CD8 cys-diabodies for immunoPET assessment of immune activation in normal mice and tumor-bearing mice treated with immune stimulants such as anti-CD137 antibodies. Specific Aim 3 will focus on immunoPET detection and quantification of CD8+T cells in tumor-bearing mice treated with antibodies that reverse immunosuppression (such as anti-PD-1 and anti-CTLA-4) and induce tumor infiltration by cytotoxic T cells. These studies will validate the role of immunoPET probes for CD8+ T cells to study responses to cancer immunotherapy in vivo, provide a deeper understanding of immune responses in living organisms, and will open the door to broad methods for imaging and quantitating immune cell subsets in preclinical models and eventually humans.